1. Field of the Invention
The present invention is directed to adenosine agonist and antagonist compounds covalently coupled to medium molecular weight and high molecular weight dextrans and microbeads. These adenosine agonist and antagonist complexes alone or in combination are able to selectively activate intravascular endothelial adenosine receptors as well as extravascular adenosine receptors.
2. Discussion of the Background
Adenosine is a low molecular weight (Mw=267) naturally occurring nucleoside having several receptor-mediated effects in mammals with potential therapeutic use. These effects of adenosine can be blocked at the receptor level by theophylline and other methylxanthines. The effects of intravascular administration of adenosine include: coronary and general vascular dilation, inhibition of the release of renin and catecholamine, auricular-ventricular blockade and spontaneous ventricular tachycardial depression and reduction of myocardial perfusion injury. Thus, adenosine can potentially be a coronary vasodilator, an anti-hypertensive agent (by causing general vascular dilation and inhibition of the release of renin and catecholamines) and an antiarrhythmic agent and a protective agent against myocardial infarction.
However, when adenosine is given intravascularly, it distributes itself throughout the entire extracellular (intravascular plus intrastitial) compartment, thereby being able to affect all cells containing adenosine receptors. Additionally, adenosine is rapidly taken up by all cells and inactivated by metabolizing enzymes. These two properties limit the therapeutic use of this nucleoside. Due to lack of compartmentalization, adenosine cannot act solely on specific target cells, but affects all cells and thereby loses specificity. Further, adenosine is rapidly metabolized so that its effects are very transitory and have no stability.
The cardiovascular actions of adenosine have been extensively studied and include coronary vasodilation, a negative chronotropic/dromotropic effect an anti-adrenergic response and cardiac protection against infarction (Olafsson et al, Circulation, 76(5):1135-1145 (1987); Babbitt et al, Circulation, 80:1388-1399 (1989); Liu et al, Circulation, 84:350-356 (1991)). While it is now known that most of these actions are mediated via membrane bound receptors, the precise mechanisms by which adenosine exerts its cardiovascular effects have yet to be defined. Difficulties arise secondary to the numerous influences involved with the formation and metabolism of adenosine. With the myocardium, adenosine can be produced and metabolized by both the endothelium and cardiomyocytes, and is therefore subject to the influence of both cell types. The relative contribution of these two cell types to the extracellular level of adenosine is dependent on several physiological factors and is still an area of controversy. There is also uncertainty as to the actual site of action of adenosine and whether or not some of its vascular effects are in part mediated via the vascular endothelium.
Endothelial dependent vascular relaxation is a well studied phenomena and has been documented for several substances including acetylcholine, ATP, ADP and substance P. For a review see Furchgott, Circ. Res., 53:557-73, 1983 and Luscher et al, CRC, Boca Raton, pp. 1-87, 1990. However, it has been difficult to establish the role of the vascular endothelium with respect to adenosine mediated vascular relaxation. For instance, studies on isolated arterial segments have shown a reduction in the vasodilatory effects of adenosine in endothelial denuded arterial segments. See Frank, G. W. and Bevan, J. A., Regulatory Function of Adenosine, Berne, R. M., Rall, T. W., Rubio, R. (eds), Martinus Nijhoff, Hague Boston London, p. 511 (1983); Haendrick, J., Berne, R. M., Am. J. Physiol., 259:H62-H67 (1990); Kennedy, C., Burnstock, G., Blood Vessels, 22:145-155 (1985); Moritoki, H., Role of Adenosine and Adenine Nucleotides in the Biological System, Imai S., Nakazawa, M. (eds), Elseveir, Amsterdam New York Oxford, pp. 217-224 (1991); Rubanyi, G. Vanhoutte, P. M., J. Cardiovasc. Pharmacol., 7:139-144 (1985). At the same time, adenosine vasodilatory effects have been reported to be independent of the vascular endothelium. See Cassis, L. A., Loeb, A. L., Peach, M. J., Topics and Perspectives in Adenosine Research, Gerlach, E., Becker, B. F. (eds), Springer, Berlin Heidelberg New York, pp. 486-496 (1987); Luscher, T. F., Vanhoutte, P.M., CRC, Boca Raton, pp. 1-87 (1990); Mathieson, J. I., Burnstock, G., Europ. J. Pharmacol., 118:221-229 (1985); Pearson, J. D., Gordon, J. L., Nature, 181:384-186 (1979). In some intact vascular beds, the endothelium may be necessary for the maximum vasodilatory response to adenosine. Nonetheless, it is not possible to assess the relative contribution of the vascular endothelium using conventional methods of comparing blood vessel responses both with and without the endothelium. This is because the vascular endothelium of intact vascular beds cannot be denuded without altering organ function. Moreover, the relative importance of the endothelium becomes evident if one considers that adenosine remains confined to the intravascular compartment during its intracoronary infusion, in up to micromolar concentrations, secondary to the impermeable metabolic barrier imposed by the endothelium (see Nees, S., Herzog, V., Becker, B. F., Bock, M., Des Rosiers, C., Gerlach, E., Basic Res. Cardiol., 80:515-529 (1985); Nees, S., Herzog, V., Becker, B. F., Bock, M., Des Rosiers, C., Gerlach, E., Adenosine: Receptors and Modulation of Cell Function, Stefanovich, V., Rudolph, K., Schubert, P. (eds), IRI, Oxford, pp. 419-436 (1985); Nees, S., Gerlach, E., Regulatory Function of Adenosine, Berne, R. M., Rall, T. W., Rubio, R. (eds), Martinus Nijhoff, Hague Boston London, pp. 347-360 (1983)) and yet the vasodilatory and negative dromotropic effects of adenosine are observable at these concentrations (Nees, S., Gerlach, E., ibid. ).
The pharmacokinetics of macromolecular adenosine analogs are similar to their low molecular weight counterparts during intracoronary infusion. Nees et al have studied the metabolic effects of perfusing isolated guinea-pig hearts with polyadenylic acid (poly-A; molecular weight: 100,000). See Nees, S., Herzog, V., Becker, B. F., Bock, M., Des Rosiers,, C., Gerlach, E., Basic. Res. Cardiol., 80:515-529 (1985). Olsson et al covalently bonded adenosine and theophylline to oxidized stachyose. Anesthetized dogs were then administered these compounds by intracoronary infusion to study dose-dependent coronary vasodilation. See Olsson, R. A., Davis, C. J., Khouri, E. M., Patterson, R. E., Cir. Res., 39:93-98 (1976). Schrader et al covalently coupled adenosine monophosphate (AMP) to the enzyme carbonic anhydrase to produce a conjugate having a mean molecular weight of about 30,000. When infused into the coronary arteries of isolated guinea-pig hearts, the conjugate induced vasodilation which was similar in magnitude and time course to the vasodilation elicited by free AMP or adenosine. See Schrader, J., Nees, S., Gerlach, E., Pfluger Arch., 369:251-257 (1977). Intracoronary infusion of adenosine deaminase, which deaminates adenosine to inosine, alters cardiovascular function and yet this enzyme remains largely intravascularly confined. Clemo, H. F., Belardinelli, L., Cir. Res., 59:437-446 (1986).
Although Schrader et al, Nees et al and Olsson et al couple adenosine to larger molecules, these derivatives are not large enough to completely prevent the passage of the derivatives through the endothelium and outside of the vascular compartment. Selective activation of intravascular adenosine receptors is not possible with these relatively low molecular weight adenosine derivatives.
There is a continuing need for adenosine agonist and antagonist compounds which are useful in studying the functional significance of intravascular, endogenous and exogenous coronary adenosine. Further, adenosine compounds which selectively activate intravascular or interstitial adenosine receptors or which block these receptors are useful pharmaceutical agents in eliciting specific cardiovascular effects in mammals.